Tenascin-C nucleic acid ligands

ABSTRACT

Methods are described for the identification and preparation of nucleic acid ligands to tenascin-C. Included in the invention are specific RNA ligands to tenascin-C identified by the SELEX method. Further included in the invention are methods for detecting the presence of a disease condition in a biological tissue in which tenascin-C is expressed.

FIELD OF THE INVENTION

Described herein are high affinity nucleic acid ligands to tenascin-C.Also described herein are methods for identifying and preparing highaffinity nucleic acid ligands to tenascin-C. The method used herein foridentifying such nucleic acid ligands is called SELEX, an acronym forSystematic Evolution of Ligands by Exponential enrichment. Furtherdisclosed are high affinity nucleic acid ligands to tenascin-C. Furtherdisclosed are RNA ligands to tenascin-C. Also included areoligonucleotides containing nucleotide derivatives chemically modifiedat the 2′-positions of the purines and pyrimidines. Additionallydisclosed are RNA ligands to tenascin-C containing 2′-F and 2′OMemodifications. The oligonucleotides of the present invention are usefulas diagnostic and/or therapeutic agents.

BACKGROUND OF THE INVENTION

Tenascin-C is a 1.1–1.5 million Da, hexameric glycoprotein that islocated primarily in the extracellular matrix. Tenascin-C is expressedduring embryogenesis, wound healing, and neoplasia, suggesting a rolefor this protein in tissue remodeling (Erickson and Bourdon (1989) AnnRev Cell Biol 5:71–92). Neoplastic processes also involve tissueremodeling, and tenascin-C is over-expressed in many tumor typesincluding carcinomas of the lung, breast, prostate, and colon,astrocytomas, glioblastomas, melanomas, and sarcomas (Soini et al.(1993) Am J Clin Pathol 100(2):145–50; Koukoulis et al. (1991) HumPathol 22(7):636–43: Borsi et al. (1992) Int J Cancer 52(5):688–92;Koukoulis et al. (1993) J Submicrosc Cytol Pathol 25(2):285–95; Ibrahimet al. (1993) Hum Pathol 24(9):982–9; Riedl et al. (1998) Dis ColonRectum 41(1):86–92; Tuominen and Kallioinen (1994) J Cutan Pathol21(5):424–9; Natali et al. (1990) Int J Cancer 46(4):586–90; Zagzag etal. (1995) Cancer Res 55(4):907–14; Hasegawa et al. (1997) ActaNeuropathol (Berl) 93(5):431–7; Saxon et al. (1997) Pediatr Pathol LabMed 17(2):259–66; Hasegawa et al. (1995) Hum Pathol 26(8):838–45). Inaddition, tenascin-C is overexpressed in hyperproliferative skindiseases, e.g. psoriasis (Schalkwijk et al. (1991) Br J Dermatol124(1):13–20), and in atherosclerotic lesions (Fukumoto et al. (1998) JAtheroscler Thromb 5(1):29–35; Wallner et al. (1999) Circulation99(10):1284–9). Radiolabeled antibodies that bind tenascin-C are usedfor imaging and therapy of tumors in clinical settings (Paganelli et al.(1999) Eur J Nucl Med 26(4):348–57; Paganelli et al. (1994) Eur J NuclMed 21(4):314–21. Bigner et al. (1998) J Clin Oncol 16(6):2202–12; Merloet al. (1997) Int J Cancer 71(5):810–6).

Aptamers against tenascin-C have potential utility for cancer diagnosisand therapy, as well as for diagnosis and therapy of atheroslerosis andtherapy of psoriasis. Relative to antibodies, aptamers are small (7–20kDa), clear very rapidly from blood, and are chemically synthesized.Rapid blood clearance is important for in vivo diagnostic imaging, whereblood levels are a primary determinant of background that obscures animage. Rapid blood clearance may also be important in therapy, whereblood levels may contribute to toxicity. SELEX technology allows rapidaptamer isolation, and chemical synthesis enables facile andsite-specific conjugation of aptamers to a variety of inert andbioactive molecules. An aptamer to tenascin-C would therefore be usefulfor tumor therapy or in vivo or ex vivo diagnostic imaging and/or fordelivering a variety of therapeutic agents complexed with the tenascin-Cnucleic acid ligand for treatment of disease conditions in whichtenascin-C is expressed.

The dogma for many years was that nucleic acids had primarily aninformational role. Through a method known as Systematic Evolution ofLigands by EXponential enrichment, termed the SELEX process, it hasbecome clear that nucleic acids have three dimensional structuraldiversity not unlike proteins. The SELEX process is a method for the invitro evolution of nucleic acid molecules with highly specific bindingto target molecules and is described in U.S. patent application Ser. No.07/536,428, filed Jun. 11, 1990, entitled “Systematic Evolution ofLigands by EXponential Enrichment,” now abandoned, U.S. Pat. No.5,475,096 entitled “Nucleic Acid Ligands,” U.S. Pat. No. 5,270,163 (seealso WO 91/19813) entitled “Methods for Identifying Nucleic AcidLigands,” each of which is specifically incorporated by reference hereinin its entirety. Each of these applications, collectively referred toherein as the SELEX patent applications, describes a fundamentally novelmethod for making a nucleic acid ligand to any desired target molecule.The SELEX process provides a class of products which are referred to asnucleic acid ligands or aptamers, each having a unique sequence, andwhich have the property of binding specifically to a desired targetcompound or molecule. Each SELEX-identified nucleic acid ligand is aspecific ligand of a given target compound or molecule. The SELEXprocess is based on the unique insight that nucleic acids havesufficient capacity for forming a variety of two- and three-dimensionalstructures and sufficient chemical versatility available within theirmonomers to act as ligands (form specific binding pairs) with virtuallyany chemical compound, whether monomeric or polymeric. Molecules of anysize or composition can serve as targets in the SELEX method. The SELEXmethod applied to the application of high affinity binding involvesselection from a mixture of candidate oligonucleotides and step-wiseiterations of binding, partitioning and amplification, using the samegeneral selection scheme, to achieve virtually any desired criterion ofbinding affinity and selectivity. Starting from a mixture of nucleicacids, preferably comprising a segment of randomized sequence, the SELEXmethod includes steps of contacting the mixture with the target underconditions favorable for binding, partitioning unbound nucleic acidsfrom those nucleic acids which have bound specifically to targetmolecules, dissociating the nucleic acid-target complexes, amplifyingthe nucleic acids dissociated from the nucleic acid-target complexes toyield a ligand-enriched mixture of nucleic acids, then reiterating thesteps of binding, partitioning, dissociating and amplifying through asmany cycles as desired to yield highly specific high affinity nucleicacid ligands to the target molecule.

It has been recognized by the present inventors that the SELEX methoddemonstrates that nucleic acids as chemical compounds can form a widearray of shapes, sizes and configurations, and are capable of a farbroader repertoire of binding and other functions than those displayedby nucleic acids in biological systems.

The basic SELEX method has been modified to achieve a number of specificobjectives. For example, U.S. patent application Ser. No. 07/960,093,filed Oct. 14, 1992, now abandoned, and U.S. Pat. No. 5,707,796, bothentitled “Method for Selecting Nucleic Acids on the Basis of Structure,”describe the use of the SELEX process in conjunction with gelelectrophoresis to select nucleic acid molecules with specificstructural characteristics, such as bent DNA. U.S. patent applicationSer. No. 08/123,935, filed Sep. 17, 1993, entitled “Photoselection ofNucleic Acid Ligands,” now abandoned, U.S. Pat. No. 5,763,177, entitled“Systematic Evolution of Ligands by Exponential Enrichment:Photoselection of Nucleic Acid Ligands and Solution SELEX” and U.S.patent application Ser. No. 09/093,293, filed Jun. 8, 1998, entitled“Systematic Evolution of Ligands by Exponential Enrichment:Photoselection of Nucleic Acid Ligands and Solution SELEX.” describe aSELEX based method for selecting nucleic acid ligands containingphotoreactive groups capable of binding and/or photocrosslinking toand/or photoinactivating a target molecule. U.S. Pat. No. 5,580,737,entitled “High-Affinity Nucleic Acid Ligands That Discriminate BetweenTheophylline and Caffeine,” describes a method for identifying highlyspecific nucleic acid ligands able to discriminate between closelyrelated molecules, which can be non-peptidic, termed Counter-SELEX. U.S.Pat. No. 5,567,588, entitled “Systematic Evolution of Ligands byEXponential Enrichment: Solution SELEX,” describes a SELEX-based methodwhich achieves highly efficient partitioning between oligonucleotideshaving high and low affinity for a target molecule.

The SELEX method encompasses the identification of high-affinity nucleicacid ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX process-identified nucleic acid ligands containingmodified nucleotides are described in U.S. Pat. No. 5,660,985, entitled“High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,”that describes oligonucleotides containing nucleotide derivativeschemically modified at the 5- and 2′-positions of pyrimidines. U.S. Pat.No. 5,580,737, supra, describes highly specific nucleic acid ligandscontaining one or more nucleotides modified with 2′-amino (2′-NH₂),2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S. patent applicationSer. No. 08/264,029, filed Jun. 22, 1994, entitled “Novel Method ofPreparation of Known and Novel 2′ Modified Nucleosides by IntramolecularNucleophilic Displacement,” now abandoned, describes oligonucleotidescontaining various 2′-modified pyrimidines.

The SELEX method encompasses combining selected oligonucleotides withother selected oligonucleotides and non-oligonucleotide functional unitsas described in U.S. Pat. No. 5,637,459, entitled “Systematic Evolutionof Ligands by EXponential Enrichment: Chimeric SELEX,” and U.S. Pat. No.5,683,867, entitled “Systematic Evolution of Ligands by EXponentialEnrichment: Blended SELEX,” respectively. These applications allow thecombination of the broad array of shapes and other properties, and theefficient amplification and replication properties, of oligonucleotideswith the desirable properties of other molecules.

The SELEX method further encompasses combining selected nucleic acidligands with lipophilic compounds or non-immunogenic, high molecularweight compounds in a diagnostic or therapeutic complex as described inU.S. patent application Ser. No. 08/434,465, filed May 4, 1995, entitled“Nucleic Acid Ligand Complexes”. Each of the above described patents andapplications which describe modifications of the basic SELEX procedureare specifically incorporated by reference herein in their entirety.

SUMMARY OF THE INVENTION

The present invention describes a method for isolating nucleic acidligands that bind to tenascin-C with high specificity. Further describedherein are nucleic acid ligands to tenascin-C. Also described herein arehigh affinity RNA ligands to tenascin-C. Further described are2′fluoro-modified pyrimidine and 2′OMe-modified purine RNA ligands totenascin-C. The method utilized herein for identifying such nucleic acidligands is called SELEX, an acronym for Systematic Evolution of Ligandsby Exponential enrichment. Included herein are the ligands that areshown in Tables 3 and 4 and FIG. 10.

Further included in this invention is a method for detecting thepresence of a disease that is expressing tenascin-C in a biologicaltissue that may contain the disease. Still further included in thisinvention is a method for detecting the presence of a tumor that isexpressing tenascin-C in a biological tissue that may contain the tumor.Further included in this invention is a complex for use in in vivo or exvivo diagnostics. Still further included in this invention is a methodfor delivering therapeutic agents for the treatment or prophylaxis ofdiseased tissues that express tenascin-C. Still further included in thisinvention is a complex for use in delivering therapeutic agents fortreatment or prophylaxis of diseased tissues that express tenascin-C.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows binding of Cell SELEX RNA pools to U251 cells.

FIG. 2 shows proposed secondary structure of aptamers TTA1 and TTA1.NB.Included in the figure is the conjugation of the aptamers with Tc-99mchelator. All A's are 2′OMe modified. All G's, except as indicated, are2′OMe modified. All C's and U's are 2′F modified.

FIG. 3 shows images of U251 tumor xenografts in mice, obtained usingTc-99 m-labeled TTA1 and TTA1.NB, three hours post-injection.

FIG. 4 shows fluorescence microscopy of a U251 glioblastoma tumorsection, taken three hours after i.v. injection ofRhodamine-Red-X-labeled TTA1.

FIG. 5 shows the way in which the Tc-99m and linker is bound through the5′G of TTA1.

FIG. 6 describes TTA1/GS7641 uptake at 3 hours into various human tumorxenografts in mouse, compared to uptake of the non-binding controlaptamer. ID/g=injected dose/gram.

FIG. 7 displays the biodistribution of In-111 labeled TTA1/GS7641 usingeither DOTA or DTPA as the radiometal chelator, 3 hours after injection.ID/g=injected dose/gram.

FIG. 8 shows the conjugation of the aptamer to DTPA. The ¹¹¹In is shownas chelated by DTPA.

FIG. 9 shows the conjugation of the aptamer to DOTA. The ¹¹¹In is shownas chelated by DOTA.

FIG. 10 shows the proposed secondary structure of aptamer TTA1. Includedin the figure is the conjugation of the aptamer with Tc-99m chelator.The aptamer is shown in its Tc-99m labled form. All A's are 2′OMemodified. All G's, except as indicated, are 2′OMe modified. All C's andU's are 2′F modified.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The central method utilized herein for identifying nucleic acid ligandsto tenascin-C is called the SELEX process, an acronym for SystematicEvolution of Ligands by Exponential enrichment and involves (a)contacting the candidate mixture of nucleic acids with tenascin-C (b)partitioning between members of said candidate mixture on the basis ofaffinity to tenascin-C, and c) amplifying the selected molecules toyield a mixture of nucleic acids enriched for nucleic acid sequenceswith a relatively higher affinity for binding to tenascin-C. Theinvention includes RNA ligands to tenascin-C. This invention furtherincludes the specific RNA ligands to tenascin-C shown in Tables 3 and 4and FIG. 10. More specifically, this invention includes nucleic acidsequences that are substantially homologous to and that havesubstantially the same ability to bind tenascin-C as the specificnucleic acid ligands shown in Tables 3 and 4 and FIG. 10. Bysubstantially homologous it is meant a degree of primary sequencehomology in excess of 70%, most preferably in excess of 80%, and evenmore preferably in excess of 90%, 95%, or 99%. The percentage ofhomology as described herein is calculated as the percentage ofnucleotides found in the smaller of the two sequences which align withidentical nucleotide residues in the sequence being compared when 1 gapin a length of 10 nucleotides may be introduced to assist in thatalignment. Substantially the same ability to bind tenascin-C means thatthe affinity is within one or two orders of magnitude of the affinity ofthe ligands described herein. It is well within the skill of those ofordinary skill in the art to determine whether a givensequence—substantially homologous to those specifically describedherein—has the same ability to bind tenascin-C.

A review of the sequence homologies of the nucleic acid ligands oftenascin-C shown in Tables 3 and 4 and FIG. 10 shows that sequences withlittle or no primary homology may have substantially the same ability tobind tenascin-C. For these reasons, this invention also includes nucleicacid ligands that have substantially the same postulated structure orstructural motifs and ability to bind tenascin-C as the nucleic acidligands shown in Tables 3 and 4 and FIG. 10. Substantially the samestructure or structural motifs can be postulated by sequence alignmentusing the Zukerfold program (see Zuker (1989) Science 244:48–52). Aswould be known in the art, other computer programs can be used forpredicting secondary structure and structural motifs. Substantially thesame structure or structural motif of nucleic acid ligands in solutionor as a bound structure can also be postulated using NMR or othertechniques as would be known in the art.

Further included in this invention is a method for detecting thepresence of a disease that is expressing tenascin-C in a biologicaltissue which may contain the disease by the method of (a) identifying anucleic acid ligand from a candidate mixture of nucleic acids, thenucleic acid ligand being a ligand of tenascin-C, by the methodcomprising (i) contacting a candidate mixture of nucleic acids withtenascin-C, wherein nucleic acids having an increased affinity totenascin-C relative to the candidate mixture may be partitioned from theremainder of the candidate mixture; (ii) partitioning the increasedaffinity nucleic acids from the remainder of the candidate mixture;

(iii) amplifying the increased affinity nucleic acids to yield a mixtureof nucleic acids with relatively higher affinity and specificity forbinding to tenascin-C, whereby a nucleic acid ligand of tenascin-C isidentified; (b) attaching a marker that can be used in in vivo or exvivo diagnostics to the nucleic acid ligand identified in step (iii) toform a marker-nucleic acid ligand complex; (c) exposing a tissue whichmay contain the disease to the marker-nucleic acid ligand complex; and(d) detecting the presence of the marker-nucleic acid ligand in thetissue, whereby a disease expressing tenascin-C is identified.

It is a further object of the present invention to provide a complex foruse in in vivo or ex vivo diagnostics comprising one or more tenascin-Cnucleic acid ligands and one or more markers. Still further included inthis invention is a method for delivering therapeutic agents for thetreatment or prophylaxis of disease conditions in which tenascin-C isexpressed. Still further included in this invention is a complex for usein delivering therapeutic agents for treatment or prophylaxis of diseaseconditions in which tenascin-C is expressed.

Definitions

Various terms are used herein to refer to aspects of the presentinvention. To aid in the clarification of the description of thecomponents of this invention, the following definitions are provided:

As used herein, “nucleic acid ligand” is a non-naturally occurringnucleic acid having a desirable action on a target. Nucleic acid ligandsare often referred to as “aptamers.” The target of the present inventionis tenascin-C, hence the term tenascin-C nucleic acid ligand. Adesirable action includes, but is not limited to, binding of the target,catalytically changing the target, reacting with the target in a waywhich modifies/alters the target or the functional activity of thetarget, covalently attaching to the target as in a suicide inhibitor,facilitating the reaction between the target and another molecule. Inthe preferred embodiment, the action is specific binding affinity for atarget molecule, such target molecule being a three dimensional chemicalstructure other than a polynucleotide that binds to the nucleic acidligand through a mechanism which predominantly depends on Watson/Crickbase pairing or triple helix binding, wherein the nucleic acid ligand isnot a nucleic acid having the known physiological function of beingbound by the target molecule. Nucleic acid ligands are identified from acandidate mixture of nucleic acids, said nucleic acid ligand being aligand of a tenascin-C, by the method comprising: a) contacting thecandidate mixture with tenascin-C, wherein nucleic acids having anincreased affinity to tenascin-C relative to the candidate mixture maybe partitioned from the remainder of the candidate mixture; b)partitioning the increased affinity nucleic acids from the remainder ofthe candidate mixture; and c) amplifying the increased affinity nucleicacids to yield a ligand-enriched mixture of nucleic acids (see U.S.patent application Ser. No. 08/434,425, filed May 3, 1995, now U.S. Pat.No. 5,789,157, which is hereby incorporated herein by reference).

As used herein, “candidate mixture” is a mixture of nucleic acids ofdiffering sequence from which to select a desired ligand. The source ofa candidate mixture can be from naturally-occurring nucleic acids orfragments thereof, chemically synthesized nucleic acids, enzymaticallysynthesized nucleic acids or nucleic acids made by a combination of theforegoing techniques. In a preferred embodiment, each nucleic acid hasfixed sequences surrounding a randomized region to facilitate theamplification process.

As used herein, “nucleic acid” means either DNA, RNA, single-stranded ordouble-stranded, and any chemical modifications thereof. Modificationsinclude, but are not limited to, those which provide other chemicalgroups that incorporate additional charge, polarizability, hydrogenbonding, electrostatic interaction, and fluxionality to the nucleic acidligand bases or to the nucleic acid ligand as a whole. Suchmodifications include, but are not limited to, 2′-position sugarmodifications, 5-position pyrimidine modifications, 8-position purinemodifications, modifications at exocyclic amines, substitution of4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbonemodifications, methylations, unusual base-pairing combinations such asthe isobases isocytidine and isoguanidine and the like. Modificationscan also include 3′ and 5′ modifications such as capping.

“SELEX” methodology involves the combination of selection of nucleicacid ligands which interact with a target in a desirable manner, forexample binding to a protein, with amplification of those selectednucleic acids. Optional iterative cycling of the selection/amplificationsteps allows selection of one or a small number of nucleic acids whichinteract most strongly with the target from a pool which contains a verylarge number of nucleic acids. Cycling of the selection/amplificationprocedure is continued until a selected goal is achieved. In the presentinvention, the SELEX methodology is employed to obtain nucleic acidligands to tenascin-C.

The SELEX methodology is described in the SELEX patent applications.

“SELEX target” or “target” means any compound or molecule of interestfor which a ligand is desired. A target can be a protein, peptide,carbohydrate, polysaccharide, glycoprotein, hormone, receptor, antigen,antibody, virus, substrate, metabolite, transition state analog,cofactor, inhibitor, drug, dye, nutrient, growth factor, etc. withoutlimitation. In this application, the SELEX target is tenascin-C.

“Complex” as used herein means the molecular entity formed by thecovalent linking of one or more tenascin-C nucleic acid ligands with oneor more markers. In certain embodiments of the present invention, thecomplex is depicted as A—B—Y, wherein A is a marker; B is optional, andcomprises a linker; and Y is a tenascin-C nucleic acid ligand.

“Marker” as used herein is a molecular entity or entities that whencomplexed with the tenascin-C nucleic acid ligand, either directly orthrough a linker(s) or spacer(s), allows the detection of the complex inan in vivo or ex vivo setting through visual or chemical means. Examplesof markers include, but are not limited to radionuclides, includingTc-99m, Re-188, Cu-64, Cu-67, F-18, ¹²⁵I, ¹³¹I, ¹¹¹In, ³²P, ¹⁸⁶Re; allfluorophores, including fluorescein, rhodamine, Texas Red; derivativesof the above fluorophores, including Rhodamine-Red-X, magneticcompounds; and biotin.

As used herein, “linker” is a molecular entity that connects two or moremolecular entities through covalent bond or non-covalent interactions,and can allow spatial separation of the molecular entities in a mannerthat preserves the functional properties of one or more of the molecularentities. A linker can also be known as a spacer. Examples of a linkerinclude, but are not limited to, the (CH₂CH₂O)₆ and hexylaminestructures shown in FIG. 2.

“Therapeutic” as used herein, includes treatment and/or prophylaxis.When used, therapeutic refers to humans and other animals.

“Covalent Bond” is the chemical bond formed by the sharing of electrons.

“Non-covalent interactions” are means by which molecular entities areheld together by interactions other than Covalent Bonds including ionicinteractions and hydrogen bonds.

In the preferred embodiment, the nucleic acid ligands of the presentinvention are derived from the SELEX methodology. The SELEX process isdescribed in U.S. patent application Ser. No. 07/536,428, entitled“Systematic Evolution of Ligands by Exponential Enrichment,” nowabandoned, U.S. Pat. No. 5,475,096, entitled “Nucleic Acid Ligands” andU.S. Pat. No. 5,270,163, (see also WO 91/19813), entitled “Methods forIdentifying Nucleic Acid Ligands.” These applications, each specificallyincorporated herein by reference, are collectively called the SELEXpatent applications.

The SELEX process provides a class of products which are nucleic acidmolecules, each having a unique sequence, and each of which has theproperty of binding specifically to a desired target compound ormolecule. Target molecules are preferably proteins, but can also includeamong others carbohydrates, peptidoglycans and a variety of smallmolecules. SELEX methodology can also be used to target biologicalstructures, such as cell surfaces or viruses, through specificinteraction with a molecule that is an integral part of that biologicalstructure.

In its most basic form, the SELEX process may be defined by thefollowing series of steps:

1) A candidate mixture of nucleic acids of differing sequence isprepared. The candidate mixture generally includes regions of fixedsequences (i.e., each of the members of the candidate mixture containsthe same sequences in the same location) and regions of randomizedsequences. The fixed sequence regions are selected either: (a) to assistin the amplification steps described below, (b) to mimic a sequenceknown to bind to the target, or (c) to enhance the concentration of agiven structural arrangement of the nucleic acids in the candidatemixture. The randomized sequences can be totally randomized (i.e., theprobability of finding a base at any position being one in four) or onlypartially randomized (e.g., the probability of finding a base at anylocation can be selected at any level between 0 and 100 percent).

2) The candidate mixture is contacted with the selected target underconditions favorable for binding between the target and members of thecandidate mixture. Under these circumstances, the interaction betweenthe target and the nucleic acids of the candidate mixture can beconsidered as forming nucleic acid-target pairs between the target andthose nucleic acids having the strongest affinity for the target.

3) The nucleic acids with the highest affinity for the target arepartitioned from those nucleic acids with lesser affinity to the target.Because only an extremely small number of sequences (and possibly onlyone molecule of nucleic acid) corresponding to the highest affinitynucleic acids exist in the candidate mixture, it is generally desirableto set the partitioning criteria so that a significant amount of thenucleic acids in the candidate mixture (approximately 5–50%) areretained during partitioning.

4) Those nucleic acids selected during partitioning as having therelatively higher affinity for the target are then amplified to create anew candidate mixture that is enriched in nucleic acids having arelatively higher affinity for the target.

5) By repeating the partitioning and amplifying steps above, the newlyformed candidate mixture contains fewer and fewer unique sequences, andthe average degree of affinity of the nucleic acids to the target willgenerally increase. Taken to its extreme, the SELEX process will yield acandidate mixture containing one or a small number of unique nucleicacids representing those nucleic acids from the original candidatemixture having the highest affinity to the target molecule.

The basic SELEX method has been modified to achieve a number of specificobjectives. For example, U.S. patent application Ser. No. 07/960,093,filed Oct. 14, 1992, now abandoned, and U.S. Pat. No. 5,707,796, bothentitled “Method for Selecting Nucleic Acids on the Basis of Structure,”describe the use of the SELEX process in conjunction with gelelectrophoresis to select nucleic acid molecules with specificstructural characteristics, such as bent DNA. U.S. patent applicationSer. No. 08/123,935, filed Sep. 17, 1993, entitled “Photoselection ofNucleic Acid Ligands,” now abandoned, U.S. Pat. No. 5,763,177, entitled“Systematic Evolution of Ligands by Exponential Enrichment:Photoselection of Nucleic Acid Ligands and Solution SELEX” and U.S.patent application Ser. No. 09/093,293, filed Jun. 8, 1998, entitled“Systematic Evolution of Ligands by Exponential Enrichment:Photoselection of Nucleic Acid Ligands and Solution SELEX,” all describea SELEX based method for selecting nucleic acid ligands containingphotoreactive groups capable of binding and/or photocrosslinking toand/or photoinactivating a target molecule. U.S. Pat. No. 5,580,737,entitled “High-Affinity Nucleic Acid Ligands That Discriminate BetweenTheophylline and Caffeine,” describes a method for identifying highlyspecific nucleic acid ligands able to discriminate between closelyrelated molecules, termed Counter-SELEX. U.S. Pat. No. 5,567,588,entitled “Systematic Evolution of Ligands by Exponential Enrichment:Solution SELEX,” describes a SELEX-based method which achieves highlyefficient partitioning between oligonucleotides having high and lowaffinity for a target molecule. U.S. Pat. No. 5,496,938, entitled“Nucleic Acid Ligands to HIV-RT and HIV-1 Rev,” describes methods forobtaining improved nucleic acid ligands after SELEX has been performed.U.S. Pat. No. 5,705,337 entitled “Systematic Evolution of Ligands byExponential Enrichment: Chemi-SELEX,” describes methods for covalentlylinking a ligand to its target.

The SELEX method encompasses the identification of high-affinity nucleicacid ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX-identified nucleic acid ligands containingmodified nucleotides are described in U.S. Pat. No. 5,660,985, entitled“High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,”that describes oligonucleotides containing nucleotide derivativeschemically modified at the 5- and 2′-positions of pyrimidines. U.S. Pat.No. 5,637,459, supra, describes highly specific nucleic acid ligandscontaining one or more nucleotides modified with 2′-amino (2′-NH₂),2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S. patent applicationSer. No. 08/264,029, filed Jun. 22, 1994, entitled “Novel Method ofPreparation of Known and Novel 2′ Modified Nucleosides by IntramolecularNucleophilic Displacement,” describes oligonucleotides containingvarious 2′-modified pyrimidines.

The SELEX method encompasses combining selected oligonucleotides withother selected oligonucleotides and non-oligonucleotide functional unitsas described in U.S. Pat. No. 5,637,459, entitled “Systematic Evolutionof Ligands by Exponential Enrichment: Chimeric SELEX,” and U.S. Pat. No.5,683,867, entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Blended SELEX,” respectively. These applications allow thecombination of the broad array of shapes and other properties, and theefficient amplification and replication properties, of oligonucleotideswith the desirable properties of other molecules.

In U.S. Pat. No. 5,496,938 methods are described for obtaining improvedNucleic Acid Ligands after the SELEX process has been performed. Thispatent, entitled “Nucleic Acid Ligands to HIV-RT and HIV-1 Rev,” isspecifically incorporated herein by reference.

U.S. patent application Ser. No. 08/434,425, entitled “SystematicEvolution of Ligands by Exponential Enrichment: Tissue SELEX,” filed May3, 1995, now U.S. Pat. No. 5,789,157, describes methods for identifyinga nucleic acid ligands to a macromolecular component of a tissue,including cancer cells, and the nucleic acid ligands so identified. Thispatent is specifically incorporated herein by reference.

One potential problem encountered in the diagnostic or therapeutic useof nucleic acids is that oligonucleotides in their phosphodiester formmay be quickly degraded in body fluids by intracellular andextracellular enzymes such as endonucleases and exonucleases before thedesired effect is manifest. Certain chemical modifications of thenucleic acid ligand can be made to increase the in vivo stability of thenucleic acid ligand or to enhance or to mediate the delivery of thenucleic acid ligand. See, e.g., U.S. patent application Ser. No.08/117,991, filed Sep. 8, 1993, now abandoned, and U.S. Pat. No.5,660,985, both entitled “High Affinity Nucleic Acid Ligands ContainingModified Nucleotides,” which is specifically incorporated herein byreference. Modifications of the nucleic acid ligands contemplated inthis invention include, but are not limited to, those which provideother chemical groups that incorporate additional charge,polarizability, hydrophobicity, hydrogen bonding, electrostaticinteraction, and fluxionality to the nucleic acid ligand bases or to thenucleic acid ligand as a whole. Such modifications include, but are notlimited to. 2′-position sugar modifications, 5-position pyrimidinemodifications, 8-position purine modifications, modifications atexocyclic amines, substitution of 4-thiouridine, substitution of 5-bromoor 5-iodo-uracil; backbone modifications, phosphorothioate or alkylphosphate modifications, methylations, unusual base-pairing combinationssuch as the isobases isocytidine and isoguanidine and the like.Modifications can also include 3′ and 5′ modifications such as capping.In preferred embodiments of the instant invention, the nucleic acidligands are RNA molecules that are 2′-fluoro (2′-F) modified on thesugar moiety of pyrimidine residues.

The modifications can be pre- or post-SELEX process modifications.Pre-SELEX process modifications yield nucleic acid ligands with bothspecificity for their SELEX target and improved in vivo stability.Post-SELEX process modifications made to 2′-OH nucleic acid ligands canresult in improved in vivo stability without adversely affecting thebinding capacity of the nucleic acid ligand.

Other modifications are known to one of ordinary skill in the art. Suchmodifications may be made post-SELEX process (modification of previouslyidentified unmodified ligands) or by incorporation into the SELEXprocess.

The nucleic acid ligands of the invention are prepared through the SELEXmethodology that is outlined above and thoroughly enabled in the SELEXapplications incorporated herein by reference in their entirety.

The tenascin-C aptamers of the invention bind to the heparin bindingsite of the tenascin-C COOH terminus.

In certain embodiments of the present invention, the nucleic acidligands to tenascin-C described herein are useful for diagnosticpurposes and can be used to image pathological conditions (such as humantumor imaging). In addition to diagnosis, the tenascin-C nucleic acidligands are useful in the prognosis and monitoring of disease conditionsin which tenascin-C is expressed.

Diagnostic agents need only be able to allow the user to identify thepresence of a given target at a particular locale or concentration.Simply the ability to form binding pairs with the target may besufficient to trigger a positive signal for diagnostic purposes. Thoseskilled in the art would be able to adapt any tenascin-C nucleic acidligand by procedures known in the art to incorporate a marker in orderto track the presence of the nucleic acid ligand. Such a marker could beused in a number of diagnostic procedures, such as detection of primaryand metastatic tumors and athersclerotic lesions. The labeling markersexemplified herein are technetium-99m and ¹¹¹In, however, other markerssuch as additional radionuclides, magnetic compounds, fluorophores,biotin, and the like can be conjugated to the tenascin-C nucleic acidligand for imaging in an in vivo or ex vivo setting disease conditionsin which tenascin-C is expressed (e.g., cancer, atherosclerosis, andpsoriasis). The marker may be covalently bound to a variety of positionson the tenascin-C nucleic acid ligand, such as to an exocyclic aminogroup on the base, the 5-position of a pyrimidine nucleotide, the8-position of a purine nucleotide, the hydroxyl group of the phosphate,or a hydroxyl group or other group at the 5′ or 3′ terminus of thetenascin-C nucleic acid ligand. In embodiments where the marker istechnetium-99m of ¹¹¹In, preferably it is bonded to the 5′ or 3′hydroxyl of the phosphate group thereof or to the 5 position of amodified pyrimidine. In the most preferred embodiment, the marker isbonded to the 5′ hydroxyl of the phosphate group of the nucleic acidligand with or without a linker. In another embodiment, the marker isconjugated to the nucleic acid ligand by incorporating a pyrimidinecontaining a primary amine at the 5 position, and use of the amine forconjugation to the marker. Attachment of the marker can be done directlyor with the utilization of a linker. In the embodiment wheretechnetium-99m or ¹¹¹In is used as the marker, the preferred linker is ahexylamine linker as shown in FIG. 10.

In other embodiments, the tenascin-C nucleic acid ligands are useful forthe delivery of therapeutic compounds (including, but not limited to,cytotoxic compounds, immune enhancing substances and therapeuticradionuclides) to tissues or organs expressing tenascin-C. Diseaseconditions in which tenascin-C may be expressed include, but are notlimited to, cancer, atherosclerosis, and psoriasis. Those skilled in theart would be able to adapt any tenascin-C nucleic acid ligand byprocedures known in the art to incorporate a therapeutic compound in acomplex. The therapeutic compound may be covalently bound to a varietyof positions on the tenascin-C nucleic acid ligand, such as to anexocyclic amino group on the base, the 5-position of a pyrimidinenucleotide, the 8-position of a purine nucleotide, the hydroxyl group ofthe phosphate, or a hydroxyl group or other group at the 5′ or 3′terminus of the tenascin-C nucleic acid ligand. In the preferredembodiment, the therapeutic agent is bonded to the 5′ amine of thenucleic acid ligand. Attachment of the therapeutic agent can be donedirectly or with the utilization of a linker. In embodiments in whichcancer is the targeted disease, 5-fluorodeoxyuracil or other nucleotideanalogs known to be active against tumors can be incorporated internallyinto existing U's within the tenascin-C nucleic acid ligand or can beadded internally or conjugated to either terminus either directly orthrough a linker. In addition, both pyrimidine analogues2′2′-difluorocytidine and purine analogues (deoxycoformycin) can beincorporated. In addition, U.S. application Ser. No. 08/993,765, filedDec. 18, 1997, incorporated herein by reference in its entirety,describes, inter alia, nucleotide-based prodrugs comprising nucleic acidligands directed to a tumor, for example tenascin-C, for preciselylocalizing chemoradiosensitizers, and radiosensitizers and radionuclidesand other radiotherapeutic agents to the tumor.

It is also contemplated that both the marker and therapeutic agent maybe associated with the tenascin-C nucleic acid ligand, such thatdetection of the disease condition and delivery of the therapeutic agentis accomplished together in one aptamer or as a mixture of two or moredifferent modified versions of the same aptamer. It is also contemplatedthat either or both the marker and/or the therapeutic agent may beassociated with a non-immunogenic, high molecular weight compound orlipophilic compound, such as a liposome. Methods for conjugating nucleicacid ligands with lipophilic compounds or non-immunogenic compounds in adiagnostic or therapeutic complex are described in U.S. patentapplication Ser. No. 08/434,465, filed May 4, 1995, entitled “NucleicAcid Ligand Complexes,” which is incorporated herein in its entirety.

The therapeutic or diagnostic compositions described herein may beadministered parenterally by injection (e.g., intravenous, subcutaneous,intradermal, intralesional), although other effective administrationforms, such as intraarticular injection, inhalant mists, orally activeformulations, transdermal iontophoresis or suppositories, are alsoenvisioned. They may also be applied locally by direct injection, can bereleased from devices, such as implanted stents or catheters, ordelivered directly to the site by an infusion pump. One preferredcarrier is physiological saline solution, but it is contemplated thatother pharmaceutically acceptable carriers may also be used. In oneembodiment, it is envisioned that the carrier and the tenascin-C nucleicacid ligand complexed with a therapeutic compound constitute aphysiologically-compatible, slow release formulation. The primarysolvent in such a carrier may be either aqueous or non-aqueous innature. In addition, the carrier may contain other pharmacologicallyacceptable excipients for modifying or maintaining the pH, osmolarity,viscosity, clarity, color, sterility, stability, rate of dissolution, orodor of the formulation. Similarly, the carrier may contain still otherpharmacologically-acceptable excipients for modifying or maintaining thestability, rate of dissolution, release, or absorption of the tenascin-Cnucleic acid ligand. Such excipients are those substances usually andcustomarily employed to formulate dosages for parental administration ineither unit dose or multi-dose form.

Once the therapeutic or diagnostic composition has been formulated, itmay be stored in sterile vials as a solution, suspension, gel, emulsion,solid, or dehydrated or lyophilized powder. Such formulations may bestored either in ready to use form or requiring reconstitutionimmediately prior to administration. The manner of administeringformulations containing tenascin-C nucleic acid ligands for systemicdelivery may be via subcutaneous, intramuscular, intravenous,intraarterial intranasal or vaginal or rectal suppository.

The following examples are provided to explain and illustrate thepresent invention and are not to be taken as limiting of the invention.Example 1 describes the materials and experimental procedures used inExample 2 for the generation of RNA ligands to tenascin-C. Example 2describes the RNA ligands to tenascin-C and the predicted secondarystructure of a selected nucleic acid ligand. Example 3 describes thedetermination of minimal size necessary for high affinity binding of aselected nucleic acid ligand, and substitution of 2′-OH purines with2′-OMe purines. Example 4 describes the biodistribution of Tc-99mlabeled tenascin-C nucleic acid ligands in tumor-bearing mice. Example 5describes the use of a fluorescently labeled tenascin-C nucleic acidligand to localize tenascin-C within tumor tissue. Example 6 describesdetection of tumors in vivo by Aptamer TTA1 (also known as GS7641).Example 7 describes alternative labeling using ¹¹¹In.

EXAMPLES Example 1 Use of SELEX to Obtain Nucleic Acid Ligands toTenascin-C and to U251 Glioblastoma Cells

Materials and Methods

Tenascin-C was purchased from Chemicon (Temecula, Calif.).Single-stranded DNA-primers and templates were synthesized by OperonTechnologies Inc. (Alameda, Calif.).

The SELEX-process has been described in detail in the SELEX PatentApplications. In brief, double-stranded transcription templates wereprepared by Klenow fragment extension of 40N7a ssDNA:5′-TCGCGCGAGTCGTCTG[40N]CCGCATCGTCCTCCC 3′ (SEQ ID NO:1)using the 5N7 primer:5′-TAATACGACTCACTATAGGGAGGACGATGCGG-3′ (SEQ ID NO:2)which contains the T7 polymerase promoter (underlined). RNA was preparedwith T7 RNA polymerase as described previously in Fitzwater and Polisky(1996) Methods Enzymol. 267: 275–301, incorporated herein by referencein its entirety. All transcription reactions were performed in thepresence of pyrimidine nucleotides that were 2′-fluoro (2′-F) modifiedon the sugar moiety. This substitution confers enhanced resistance toribonucleases that utilize the 2′-hydroxyl moiety for cleavage of thephosphodiester bond. Specifically, each transcription mixture contained3.3 mM 2′-F UTP and 3.3 mM 2′-F CTP along with 1 mM GTP and ATP. Theinitial randomized RNA library thus produced comprised 3×10¹⁴ molecules.The affinities of individual ligands for tenascin-C were determined bystandard methods using nitrocellulose filter partitioning (Tuerk andGold (1990) Science 249(4968):505–10).

For each round of SELEX, Lumino plates (Labsystems, Needham Heights,Mass.) were coated for 2 hours at room temperature with 200 μlDulbecco's PBS containing tenascin-C concentrations as shown in Table 1.After coating, wells were blocked using HBSMC+buffer [20 mM Hepes, pH7.4, 137 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂ and 1 g/liter human serumalbumin (Sigma, fraction V) for rounds 1 to 6 while for rounds 7 and 8wells were blocked HBSMC+buffer containing 1 g/liter casein (1-block;Tropix). Binding and wash buffer consisted of HBSMC+buffer containing0.05% Tween 20. For each SELEX round, RNA was diluted into 100 μl ofbinding buffer and allowed to incubate for 2 hours at 37° C. in theprotein coated wells that were pre-washed with binding buffer. Afterbinding, six washes of 200 μl each were performed. Following the washstep, the dry well was placed on top of a 95° C. heat block for 5minutes. Standard AMV reverse transcriptase reactions (50 μl) wereperformed at 48° C. directly in the well and the reaction productsutilized for standard PCR and transcription reactions. Two syntheticprimers 5N7 (see above) and 3N7a:5′-TCGCGCGAGTCGTCTG3′ (SEQ ID NO:3)were used for these template amplification and reverse transcriptionsteps.

For cell SELEX, U251 human glioblastoma cells (Hum. Hered. (1971)21:238) were grown to confluence in Dulbecco's Modified Eagle's Mediumsupplemented with 10% fetal calf serum (GIBCO BRL, Gaithersburg, Md.) onsix-well tissue culture plates (Becton Dickinson Labware, Lincoln Park,N.J.) and washed three times using Dulbecco's PBS supplemented withCaCl₂ (DPBS, GIBCO BRL) buffer. RNA labeled internally by transcription(Fitzwater (1996) supra) was incubated with the cells at 37° C. for onehour. The labeled RNA was then removed, and the cells were washed sixtimes for ten minutes each at 37° C. with DPBS. DPBS containing 5 mMEDTA was then added and incubated with the cells for 30 minutes to elutebound RNAs that remained after the washing steps. This RNA wasquantitated by a standard liquid scintillation counting protocol andamplified using RT-PCR.

Binding assays for the U251 cells. Internally labeled RNA was incubatedat increasing concentrations with confluent U251 cells in six-welltissue culture plates (Becton Dickinson Labware, Lincoln Park, N.J.) at37° C. for 60 min. Unbound RNA was washed away using three 10 minutewashes with DPBS+CaCl₂ at 37° C., and bound RNA was collected bydisrupting the cells using Trizol (Gibco BRL, Gaithersburg, Md.). BoundRNA was quantitated by liquid scintillation counting.

Cloning and Sequencing. Amplified affinity enriched oligonucleotidepools were purified on an 8% polyacrylamide gel, reverse transcribedinto ssDNA and the DNA amplified by the polymerase chain reaction (PCR)using primers containing BamH1 and HindIII restriction endonucleasesites. PCR fragments were cloned, plasmids prepared and sequenceanalyses performed according to standard techniques (Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual, 2^(nd) Ed. 3 vols., ColdSpring Harbor Laboratory Press, Cold Spring Harbor).

Example 2 RNA Ligands to Tenascin-C

Nucleic Acid Ligands to U251 cells were obtained by the SELEX processand are described in U.S. patent application Ser. No. 08/434,425,entitled “Systematic Evolution of Ligands by Exponential Enrichment:Tissue SELEX,” filed May 3, 1995, now U.S. Pat. No. 5,789,157.Subsequently it was determined that the ligands that were obtained weretenascin-C nucleic acid ligands.

To obtain oligonucleotide ligands against human tenascin-C, eight roundsof SELEX were performed using the randomized nucleotide library asdescribed above in Materials and Methods. RNA and protein input intoeach round is shown in Table 1. After 8 rounds of SELEX, the affinity ofthe oligonucleotide pool for tenascin-C was 10 nM, and this affinity didnot increase with additional SELEX rounds.

To obtain ligands to U251 glioblastoma cells, nine rounds of SELEX wereperformed using the randomized nucleotide library. After nine rounds ofbinding to U251 cells and EDTA elution, rounds 3, 5 and 9 were testedfor their ability to bind to U251 cells. FIG. 1 shows that as the numberof SELEX rounds increases, the amount of bound RNA also increases at aparticular concentration. Because of the complexity of the targettissue, it was not possible to estimate the affinity of theoligonucleotide pools for the unknown target molecules(s) on thesecells.

The E9 pool (nine rounds of binding and EDTA elution from U251 cells)was then used as a starting point for a SELEX against purifiedtenascin-C. Two rounds of SELEX using purified tenascin-C were performedas described above. Input protein and RNA concentrations for two roundsof SELEX (E9P1 and E9P2) are described in Table 2.

In summary, three different SELEX experiments were performed: anexperiment using purified tenascin-C as the target, an experiment usingU251 glioblastoma cells as the target, and an experiment in which theSELEX pool from the U251 glioblastoma cells was used to initiate a SELEXexperiment using purified tenascin-C as the target.

All three SELEX experiments were analyzed by cloning and sequencingligands from round 8 of the purified tenascin-C SELEX (“TN” sequences),from round 9 of the U251 cell SELEX (“E9” sequences), and from round 2of the U251/tenascin-C hybrid SELEX (“E9P2” sequences). The sequences of34 unique clones are shown in Table 3, and are divided into two majorgroups: tenascin-C ligands (“TN” and “E9P2” sequences) and U251 cellligands (“E9” ligands). Among the tenascin-C ligands, the majority ofthe clones (65 total) represent one of two distinct sequence classesdesignated Family I and Family II (FIG. 1). Examination of the variableregion of the 12 clones in Family I revealed 7 unique sequences that arerelated through the consensus sequence GACNYUUCCNGCYAC (SEQ ID NO:12).Examination of the variable region of the 18 clones in Family IIrevealed sequences that share a consensus sequence CGUCGCC (Table 3).The E9 sequences could be grouped into a related set by virtue ofconserved GAY and CAU sequences within the variable regions. Theremaining sequences did not appear related to other sequences and wereclassified as orphans. Three sequences predominate, with E9P2-1, E9P2-2,and TN9 represented 14,16, and 10 times respectively. In the “Orphan”category, one sequence, TN18, was represented twice. Overall, these datarepresent a highly enriched sequence pool.

Most individuals displayed low nanomolar dissociation constants, withthe three most prevalent sequences, TN9 and E9P2-1 and -2, having thehighest affinities at 5 nM. 2 nM, and 8 nM. (Table 3). These resultsindicate that the U251 cell SELEX is a repository for aptamers againsttenascin-C, and that only two rounds of SELEX were required to isolatethe tenascin-specific ligands from the cell SELEX pool. Oligonucleotideligands against other proteins can be similarly isolated from the E9pool using purified protein targets.

Example 3 Determination of Minimal Size of TN9, and Substitution of2′-OH Purines with 2′-OMe Purines: Synthesis of Aptamer TTA1.

Oligonucleotide synthesis procedures were standard for those skilled inthe art (Green et al. (1995) Chem Biol 2(10):683–95). 2′-fluoropyrimidine phosphoramidite monomers were obtained from JBL Scientific(San Luis Obispo, Calif.); 2′-OMe purine, 2′-OH purine, hexylamine, and(CH₂CH₂O)₆ monomers, along with the dT polystyrene solid support, wereobtained from Glen Research (Sterling, Va.). Aptamer affinities weredetermined using nitrocellulose filter partitioning (Green et al.,supra).

TN9 was chosen for further analysis based on its high affinity fortenascin-C. We first searched for a minimal sequence necessary for highaffinity binding. Using standard techniques (Green et al, supra), it wasdiscovered that nucleotides 3′ of nucleotide 55 were required forbinding to tenascin-C, while no nucleotides could be removed from the 5′end without loss of affinity. To further decrease the TN9's length from55 nucleotides and retain high affinity binding, we then attempted todefine internal deletions of TN9. The first 55 nucleotides of TN9, alongwith the first 55 nucleotides of related family II ligands TN7, TN21,and TN41, were input into a computer algorithm to determine possible RNAsecondary structure foldings (mfold 3.0, M. Zuker, D. H. Mathews & D. H.Turner. Algorithms and Thermodynamics for RNA Secondary StructurePrediction: A Practical Guide. In: RNA Biochemistry and Biotechnology,J. Barciszewski & B. F. C. Clark, eds., NATO ASI Series, Kluwer AcademicPublishers, (1999)). Among many potential RNA foldings predicted by thealgorithm, a structure common to each oligonucleotide was found. Thisstructure, represented by oligonucleotide TTA1 in FIG. 2, contains threestems that meet at a single junction, a so-called 3-stem junction. Thisfolding places the most highly conserved nucleotides of family IIoligonucleotides at the junction area. In comparing TN9, TN7, TN21, andTN41, the second stem was of variable length and sequence, suggestingthat extension of the second stem is not required for binding totenascin-C. Testing this hypothesis on TN9, we found that nucleotides10–26 could be replaced with an ethylene glycol linker, (CH₂CH₂O)₆. Thelinker serves as a substitute loop and decreases the size of theaptamer. Additionally, four-nucleotide loops (CACU or GAGA) that replacenucleotides 10–26 produce sequences with high affinity for tenascin-C.It would be well within one skilled in the art to determine othernucleotide loops or other spacers that could replace nucleotides 10–26to produce sequences with high affinity for tenascin-C.

To increase protection against nuclease activity, purine positions thatcould be substituted with the corresponding 2′-OMe purines were located.The oligonucleotide was arbitrarily divided into five sectors and allpurines within each sector were substituted by the corresponding 2′-OMepurine nucleotide, a total of five oligonucleotides (Table 4, Phase Isyntheses). The affinity of each oligonucleotide for tenascin-C wasdetermined, and it was found that all purines within sectors 1.3 and 5could be substituted without appreciable loss in affinity. Withinsectors 2 and 4, individual purines were then substituted with 2′-OMepurines and the effect of affinity was measured (Table 4, Phase IIIsyntheses). From these experiments, it was deduced that substitution ofnucleotides G9, G28, G31, and G34, with 2′OMe G causes loss in affinityfor tenascin-C. Therefore these nucleotides remain as 2′-OH purines inthe aptamer TTA1.

The aptamer TTA1 (Table 4) was then synthesized with the (CH₂CH₂O)₆(Spacer 18) linker, a 3′–3′ dT cap for exonuclease protection, a 5′hexylamine (Table 4), and all purines as 2′-OMe except the 5 Gsindicated in Table 4. A non-binding control aptamer, TTA1.NB, wasgenerated by deleting 5 nucleotides at the 3′ end to produce TTA1.NB.TTA1 binds to tenascin-C with an equilibrium dissociation constant(K_(d)) of 5 nM, while TTA1.NB has a K_(d) of >5 μM for tenascin-C.

Nucleotides 10–26 can be replaced by a non-nucleotide ethylene glycollinker. It is therefore likely that TTA1 can be synthesized in twoseparate pieces, where a break is introduced at the position of theethylene glycol linker and new 5′ and 3′ ends are introduced. Subsequentto synthesis, the two molecules will incubated together to allow hybridformation. This method allows introduction of additional amine groups aswell as nucleotides at the new 5′ and 3′ ends. The new functionalitiescould be used for bioconjugation. In addition, two-piece synthesisresults in increased chemical synthetic yield due to shortening thelength of the molecules.

Example 4 Biodistribution of Tc-99m Labeled Aptamers in Tumor-BearingMice

Aptamer biodistribution was tested by conjugating a Tc-99m chelator(Hi₁₅: Hilger et al. (1998) Tet Lett 39:9403–9406) to the 5′ end of theoligonucleotide as shown in FIG. 2, and radiolabeling the aptamer withTc-99m. The aptamer in its Tc-99m labeled form is shown in FIG. 10. TTA1and TTA1.NB were conjugated to Hi₁₅ at 50 mg/ml aptamer in 30%dimethylformamide with 5 molar equivalents of Hi₁₅—N-hydoxysuccinimide,buffered in 100 mM Na Borate pH 9.3, for 30 minutes at room temperature.The aptamer in its Tc-99m labeled form is shown in FIG. 10. Reversedphase HPLC purification yielded Hi₁₅-TTA1 and Hi₁₅-TTA1.NB. Theoligonucleotides were then labeled with Tc-99m in the following manner:to 1 nmole Hi15-aptamer was added 200 μL of 100 mM sodium phosphatebuffer, pH 8.5, 23 mg/mL NaTartrate, and 50 μL Tc-99m pertechnetate (5.0mCi) eluted from a Mo-99 column (Syncor, Denver) within 12 hours of use.The labeling reaction was initiated by the addition of 10 μL 5 mg/mLSnCl₂. The reaction mixture was incubated for 15 minutes at 90° C. Thereaction was separated from unreacted Tc-99m by spin dialysis through a30,000 MW cut-off membrane (Centrex, Schleicher & Scheull) with two 300μL washes. This labeling protocol results in 30–50% of the added 99mTcbeing incorporated with a specific activity of 2–3 mCi/nmole RNA. TheTc-99m is bound through the 5′G as shown in FIG. 5.

For biodistribution experiments, U251 xenograft tumors were prepared asfollows: U251 cells were cultured in Dulbeccos' Modified Eagle's Mediumsupplemented with 10% v/v fetal calf serum (Gibco BRL, Gaithersburg,Md.). Athymic mice (Harlan Sprague Dawley, Indianapolis, Ind.) wereinjected subcutaneously with 1×10⁶ U251 cells. When the tumors reached asize of 200–300 mg (1–2 weeks), Tc-99m labeled aptamer was injectedintravenously at 3.25 mg/kg. At indicated times, animals wereanesthetized using isoflurane (Fort Dodge Animal Health, Fort Dodge,Iowa), blood was collected by cardiac puncture, and the animal wassacrificed and tissues were harvested. Tc-99m levels were counted usinga gamma counter (Wallac Oy, Turku, Finland). Aptamer uptake into tissueswas measured as the % of injected dose per gram of tissue (% ID/g).

Images of mice were obtained using a gamma camera. Mice were placed ontothe camera (Siemens, LEM+) under anesthesia (isoflurane). Data werecollected (30 sec to 10 minutes) and analyzed using Nuclear MAC softwareversion 3.22.2 (Scientific Imaging. CA) on a Power MAC G3 (AppleComputer, CA).

Biodistribution experiments. Table 5, indicated rapid and specificuptake of the aptamer into tumor tissue; the non-binding aptamer doesnot remain in the tumor. Blood levels of Tc-99m also cleared rapidly.After three hours. Tc-99m levels brought into the tumor using Hi₁₅-TTA1had a very long half life (>18 hrs). This indicates that once theaptamer penetrates the tumor, the radiolabel carried with it remains inthe tumor for long periods of time. Such data indicate that cytotoxicagents, including radionuclides and non-radioactive agents, conjugatedto the aptamer will also remain in the tumor with long half lives.

Tc-99m radioactivity also appears in other tissues, notably the smalland large intestines. The hepatobiliary clearance pattern seen here canbe readily altered by those skilled in the art, for example by alteringthe hydrophilicity of the Tc-99m chelator, changing the chelator, orchanging the radiometal/chelator pair altogether.

Whole animal images were obtained using Tc-99m labeled Hi₁₅-TTA1 and at3 hours post-injection. Images obtained from mice injected withHi₁₅-TTA1, but not from mice injected with Hi₁₅-TTA1.NB, clearly showthe tumor (FIG. 3). Additional radioactivity is evident ingastrointestinal tract, as predicted by the biodistribution experiments.

Example 5 Use of Fluorescently Labeled TTA1 to Localize Tenascin-CWithin Tumor Tissue

Materials and Methods.

TTA1 and TTA1.NB were synthesized as described above. SuccinimdylRhodamine-Red-X (Molecular Probes, Eugene, Oreg.) was conjugated to the5′ amine of the aptamers as described above for H₁₅-NHS conjugation. TheRhodamine-Red-X-conjugated aptamers, TTA1-Red and TTA1.NB-Red, werepurified by reversed phase HPLC. U251 cell culture and tumor growth innude mice were as described above. Five nmol of TTA1-Red or TTA1.NB-Redwere injected intravenously into nude mice and at the desired time theanimal was placed under anesthesia, perfused with 0.9% NaCl, andsacrificed. The tumor was excised and placed in formalin. After 24 hr informalin, 10 μM sections were cut and Rhodamine-Red-X was detected usinga fluorescence microscope (Eclipse E800, Nikon, Japan).

Results: TTA1-Red has identical affinity for tenascin-C as theunconjugated parent aptamer, TTA1 at 5 nM. We compared tumorfluorescence levels of TTA1-Red and TTA1.NB-Red 10 min post-injection.The binding aptamer, TTA1-Red, strongly stains the tumor but notadjacent tissue (FIG. 4). In contrast, only tissue auto-fluorescence isdetected with TTA1.NB-Red. These results demonstrate the utility of theaptamer in fluorescent detection of tenascin-C in vivo, and the aptamermay be similarly used for staining tissues sections ex vivo.

Example 6 Detection of Tumors In Vivo by Aptamer TTA1 (Now Also Known asGS7641): Additional Tumor Types.

Aptamer labeling, biodistribution, and nude mouse tumor xenografts wereperformed as described in Example 4.

Many human tumor types are known to express tenascin-C. To assess theability of TTA1/GS7641 to target tumor types in addition toglioblastomas, human tumor cell lines were grown as tumors in nude mice.Tumor tissue was tested for expression of human tenascin-C, and thosetumors expressing human tenascin-C were tested for aptamer uptake. FIG.6 demonstrates aptamer uptake in several tumors, including glioblastoma,breast, colorectal, and rhabdomyosarcoma. Specific uptake into the tumoris demonstrated by the comparison between binding (TTA1/GS7641) andnon-binding aptamer (TTA1.NB). Note that KB, a xenograft that expressesmouse but not human TN-C, does not show tumor uptake. This experimentextends the observation of glioblastoma uptake into additionalcarcinomas and sarcomas, and further indicates that all tumorsexpressing human tenascin-C show uptake of TTA1/GS7641.

Example 7 Alternative Labeling Using In-111

Tumor xenograft and biodistribution studies were performed as describedin Example 4. To couple DTPA and DOTA to TTA1/GS7641, the cyclicanhydride of each was incubated with the amine-containing TTA1/GS7641under neutral pH conditions using standard methods. The structures ofthe DTPA and conjugates are shown in FIG. 8 and FIG. 9, respectively,where each has been labeled with In-111. The DOTA conjugate has theidentical linkers as for DTPA. DOTA- and DTPA-conjugates were labeledwith In-111 by incubation at 95° C. in 0.5 M NaOAc, pH 5.5 for 30 min.After removal of unincorporated radiolabel by spin dialysis over a 30 Kcut-off membrane, radiolabeled aptamer was transferred intophosphate-buffered saline for injection into tumor-bearing mice.

The biodistribution of In-11 labeled aptamer is markedly different fromthe Tc-labeled formulation described in Example 4. FIG. 7 shows that,relative to Tc-99m-labeled TTA1/GS7641, In-111-labeled TTA1/GS7641radioactivity in the intestines is greatly reduced, with a concomitantincrease in liver and kidney uptake. This experiment indicates that thechemical properties of the chelator have a large effect on distributionof the radiolabel of TTA1/GS7641 within a living animal. Biodstributionpatterns that are different from that of Hi15-TTA1/GS7641 may useful fortargeting tumors under certain clinical conditions where hepatobiliaryclearance is undesired. Such conditions include but are not limited toradiotherapy applications as well as imaging of intestines, prostate andother abdominal regions.

TABLE 1 Tenascin-C SELEX RNA and protein input. Tenascin-C RNA Round(pMol/well) (pMol/well) 1 12 200 2 12 200 3 12 200 4 12 200 5 2 33 6 233 7 2 33 8 0.2 3.3 @

TABLE 2 Cell SELEX/tenascin-C SELEX RNA and protein input Tenascin-C RNARound (pMol/well) (pMol/well) E9P1 2 33 E9P2 2 33

TABLE 3 Tenascin-C Sequences: purified protein SELEX (tenascinsequences) and U251 cell SELEX + purified protein SELEX (E9P2 sequences)SEQ ID NO: Family I TN11 4 ggGAggAcGauGcgg CAAUcAAAACUcACGUUA UUCCCUCAUCUAUUAGCUUCCC cagacgacucgcccga 10 nM TN45 5 gggaggacgaugcggCAAUCUcCGAAAAAGACUCUUCCU GCAUCCUCUcACCCCC cagacgacucgcccga 30 nM TN4 6gggaggacgaugcgg CAACCUc GAAAGACUUUUCCC GCAUCACUGUGUACUCCCCcagacgacucgcccga 40 nM TN22 7 gggaggacgaugcgg CAACCUc GAUAGACUUUUCCCGCAUCACUGUGUACUCCCC cagacgacucgcccga 40 nM TN32(2) 8 gggAggAcGauGcggcAaCCUcAA UCUuGaCAUUUCCC GcACCUAAAUUUG CCCC cagacgacucgcccga 15 nM TN149 gggaggacgaugcgg CAAACGAUC ACU UACCUUUCCU GCAUCUGCUAGC CUCCCCcagacgacucgcccga 20 nM TN44(3) 10 gggaggacgaugcggACGCCAGCCAUUGACCCUCGCUUCCACUAUUCCAUCCCCC cagacgacucgcccga 10 nM TN29(2)11 gggaggacgaugcgg CCAACCUCAUUUUGACACUUCGCCGCACCUAAUUGCCCCcagacgacucgcccga 25 nM consensus: 12 GACNYUUCCN GCAYC Family IIE9P2-4(5) 13 gggaggacgaugcgg AACCCAUA ACGCGA ACCGACCAACAUGCCUCCCGUGCCCCcagacgacucgcccga E9P2-1(14) 14 gggAggacgaugcgg UGCCCAUAGAAGCGU GCCGCUAAUGCUAACGCCCUCCCC cagacgacucgcccga 2 nM E9P2-2(16) 15gggaggacgaugcgg UGCCCACU AUGCGU GCCGAAAAACAUUUCCCCCUCUACCCcagacgacucgcccga 8 nM TN7(3) 16 gggaggacgaugcgg AACACUUUCCCAUGCGUCGCCAUACC GGAUAUAUUGCUCC cagacgacucgcccga 20 nM TN21(4) 17 gggaggacgaugcggACUGGACCAAACCGUCGCCGAUACCCGGAUACUUUGCUCC cagacgacucgcccga 10 nM TN9(10)18 gggaggacgaugcgg AACAAUGCACUCGUCGCCGUAAU GGAUGUUUUGCUCCCUGcagacgacucgcccga 5 nM TN41 19 gggaggacgaugcgg UUAAGUCUCGGUUGAAUGCCCAUCCC AGAUCCCCCUGACC cagacgacucgcccga 20 nM consensus: GCGUCGCCGOrphans E9P2-17 20 gggaggacgaugcggAUGGCAAGUCGAACCAUCCCCCACGCUUCUCCUGUUCCCC cagacgacucgcccga E9P2-48 21gggaggacgaugcgg GAAGUUUUcUCUGCCUUGGUUUCGAUUGGCGCCUccCCCCcagacgacucgcccga1 E9P2-14 22 gggaggacgaugcggUCGAGCGgUCGACCGUCAACAAGAAUAAAGCGUGUCCCUG cagacgacucgcccga E9P2-17 23gggaggacgaugcgg AUGGCAAGUCGAACCAUCCCCCACGCUUCUCCUGUUCCCCcagacgacucgcccga E9P2-22 24 gggaggacgaugcggACUAGACcgCGAGUCCAUUCAACUUGCCCAAAAaAAAACcUCCCC cagacgacucgcccga E9P2-4025 gggaggacgaugcgg GAGAUCAACAUUCCUCUAGUUUGGUUCCAACCUACACCCCcagacgacucgcccga E922-41 26 gggaggacgaugcggACGAGCGUCUCAUGAUCACACUAUUUCGUCUCAGUGUGCA cagacgacucgcccga TN18 27gggaggacgaugcgg UCGACCUCGAAUGACUCUCCACCUAUCUAACAUCCCCCCCcagacgacucgcccga 145 nM TN20 28 gggaggacgaugcggUCGACCUCGAAUGACUCUCCACCUAUCUAACAGCCUUCCC cagacgacucgcccga TN51 29gggaggacgaugcgg ACAACUCAUCCUAACCGCUCUAACAAAUCUUGUCCGACCGcagacgacucgcccga TN8 30 gggaggacgaugcggAUAAUUcGACACCAACCAGGUCCCGGAAAUCAUCCCUCUG cagacgacucgcccga >10 uM TN27 31gggaggacgaugcgg AAACCAACCGUUGACCAC CUUUUCGUUUCCGGAAAGUCCCcagacgacucgcccga 110 nM TN39 32 gggaggacgaugcggAAGCCAACCCUCUAGUCAGCCUUUCGUUUCCCACGCCACC cagacgacucgcccga TN24 33gggaggacgaugcgg gACCAACUAAACUGUUCGAAAGCUGGaACAUGUCCUGACGCcagacgacucgcccga 10 nM TN5 34 gggaggacgaugcggACCAACUAAACUGUUCGAAAGCUGGAACACGUCCUGACGC cagacgacucgcccga TN36 35gggaggacgaugcgg ACCAACUAAACUGUUCGAAAGCUAGAACACGUCCAGACGCcagacgacucgcccga TN6 36 gggaggacgaugcggACCAACUAAACUGUUCGAAAGCUGGAACACGUUCUGACGC cagacgacucgcccga TN10 37gggaggacgaugcgg ACCAACUAAACUGUUCGAAAGCUGGAAUACGUCCUGACGCcagacgacucgcccga TN1 38 gggaggacgaugcgg AAGUUUAGuGCUCCAGUUCCGACACUCCUcUACUCAGCCC cagacgacucgcccga >10 uM TN109 39gggaggacgaugcgG AgCCAGAGCCUcUcUcAGUUcUaCAGAACUuACCcACUGGcagacgacucgcccga TN110 40 gggaggacgaugcggACCUAACUCAAUCAGGAACCAAACCUAGCACUCUCAUGGC cagacgacucgcccga U251 SELEXAptamers, EDTA Elution (E9) E9-8(3) 41 gggaggacgaugcggGAGAUCAACAUUCCUCUAGUUUGGUUCCAACCUACACCCC cagacgacucgcccga E9-15 42gggaggacgaugcgg AUCUCGAUCCUUCAGCACUUCAUUUCAUUCCUUUcUGCCCcagacgacucgcccga E9-6 43 gggaggacgaugcgg ACGAUCCUUUCCUUAACAUUUCAUCAUUUCUCUUGUGCCC cagacgacucgcccga E9-5(2) 44 gggaggacgaugcggUGACGACAACUCGACUG CAUAUCUCACAACUCCUGUGCCC cagacgacucgcccga E9-3(6) 45gggaggacgaugcgg ACUAGACCGCGAGUC CAUUCAACUUGCCCAAAAACCUCCCCcagacgacucgcccga E9-9 46 gggaggacgaugcggGCGCAUCGAGCAACAUCCGAUUCGGAUUCCUCCACUCCCC cagacgacu gcccga

TABLE 4 2′-OMe Substitutions, Internal Deletions, TTA1, and TTA1.NB SEQSequence ID NO: Kd. Phase I. 2′-OMe, Affinity. TN9.3 47gggaggacgaugcggAACAAUGCACUCGUCGCCGUAAUGGAUGUUUUGCU5 >10 uM TN9.4 48GGGAGGACGAUGCGGAACAAUGCACUCGUCGCCGUAAUGGAUGUUUUGCUCCCUG5 2 nM TN9.4M1 4966676GACGAUGCGGAACAAUGCACUCGUCGCCGUAAUGGAUGUUUUGCUCCCU65 6 nM TN9.4M2 50GGGAG67C67U6C6GAACAAUGCACUCGUCGCCGUAAUGGAUGUUUUGCUCCCUG5 20 nM TN9.4M351 GGGAGGACGAUGCG677C77U6C7CUCGUCGCCGUAAUGGAUGUUUUGCUCCCUG5 7 nM TN9.4M452 GGGAGGACGAUGCGGAACAAUGCACUC6UC6CC6UAAUGGAUGUUUUGCUCCCUG5 nb TN9.4M553 GGGAGGACGAUGCGGAACAAUGCACUCGUCGCCGU77U667U6UUUU6CUCCCUG5 4 nM TN9.4Me54 6667667C67U6C6677C77U6C7CUC6UC6CC6U77U667U6UUUU6CUCCCU65 10 nM 6 =mG; 7 = mA; 5 = 3′-3′Cap, 1 = hexylamine Phase III. 2′-OMe, Affinity.TN9.4M1235 55 16667667C67U6C6677C77U6C7CUCGUCGCCGU77U667U6UUUU6CUCCCU6516.5 nM TN9.4M135G6 561666766ACGAUGCG677C77U6C7CUCGUCGCCGU77U667U6UUUU6CUCCCU65 2.2 nMTN9.4M135A7 57 166676G7CGAUGCG677C77U6C7CUCGUCGCCGU77U667U6UUUU6CUCCCU651.7 nM TN9.4M135G9 58166676GAC6AUGCG677C77U6C7CUCGUCGCCGU77U667U6UUUU6CUCCCU65 7.7 nMTN9.4M135A10 59166676GACG7UGCG677C77U6C7CUCGUCGCCGU77U667U6UUUU6CUCCCU65 1.3 nMTN9.4M135G12G14 60166676GACGAU6C6677C77U6C7CUCGUCGCCGU77U667U6UUUU6CUCCCU65 2.5 nMTN9.4M135G28 61166676GACGAUGCG677C77U6C7CUC6UCGCCGU77U667U6UUUU6CUCCCU65 37 nMTN9.4M135G31 62166676GACGAUGCG677C77U6C7CUCGUCGCCGU77U667U6UUUU6CUCCCU65 55 nMTN9.4M135G34 63166676GACGAUGCG677C77U6C7CUCGUCGCCGU77U667U6UUUU6CUCCCU65 7 nM TTA1: 645′-1G667667CG-(CH₂CH₂O)₆-CGUCGCCGU77U667U6UUUU6CUCCCU65 5 nM TTA1.NB: 655′-1G667667CG-(CH₂CH₂O)₅-CGUCGCCGU77U667U6UUUU6CU5 >5 uM

TABLE 5 Biodistribution of Tc-99m-TTA1 and -TTA1.NB min TTA1 TTA1.NB minTTA1 TTA1.NB tumor 2 4.470 ± 0.410 4.510 ± 0.300 kidney 2 44.430 ±4.280  54.470 ± 1.210  10 5.940 ± 0.590 3.020 ± 0.210 10 18.810 ± 0.940 14.320 ± 2.080  60 2.689 ± 0.310 0.147 ± 0.018 60 1.514 ± 0.040 0.637 ±0.111 180 1.883 ± 0.100 0.043 ± 0.004 180 0.286 ± 0.028 0.221 ± 0.021570 1.199 ± 0.066 0.018 ± 0.001 570 0.140 ± 0.006 0.100 ± 0.013 10201.150 ± 0.060 N/A 1020 0.081 ± 0.005 N/A blood 2 18.247 ± 1.138  15.013± 0.506  sm. int. 2 3.690 ± 0.250 3.120 ± 0.100 10 2.265 ± 0.245 2.047 ±0.195 10 7.010 ± 0.070 6.440 ± 0.250 60 0.112 ± 0.003 0.102 ± 0.019 6015.716 ± 2.036  14.649 ± 0.532  180 0.032 ± 0.001 0.034 ± 0.003 1801.479 ± 0.710 1.243 ± 0.405 570 0.013 ± 0.001 0.011 ± 0.001 570 0.219 ±0.147 0.159 ± 0.067 1020 0.006 ± 0.001 N/A 1020 0.280 ± 0.243 N/A lung 28.970 ± 1.210 8.130 ± 0.960 lg. int. 2 2.340 ± 0.240 2.280 ± 0.180 102.130 ± 0.080 1.940 ± 0.230 10 0.890 ± 0.040 0.770 ± 0.070 60 0.157 ±0.011 0.120 ± 0.005 60 10.799 ± 5.381  21.655 ± 11.676 180 0.048 ± 0.0060.041 ± 0.003 180 26.182 ± 7.839  18.023 ± 3.485  570 0.028 ± 0.0060.017 ± 0.002 570 1.263 ± 0.706 0.716 ± 0.179 1020 0.007 ± 0.001 N/A1020 0.298 ± 0.167 N/A liver 2 9.120 ± 0.530 7.900 ± 0.350 muscle 21.270 ± 0.130 1.490 ± 0.050 10 12.460 ± 1.250  9.100 ± 0.830 10 0.870 ±0.090 1.840 ± 1.000 60 1.234 ± 0.091 0.423 ± 0.095 60 0.064 ± 0.0030.050 ± 0.004 180 0.401 ± 0.084 0.211 ± 0.059 180 0.016 ± 0.002 0.011 ±0.001 570 0.104 ± 0.017 0.058 ± 0.003 570 0.011 ± 0.002 0.007 ± 0.0011020 0.075 ± 0.003 N/A 1020  0.003 ± 0.0003 spleen 2 5.100 ± 0.410 4.860± 0.130 10 2.460 ± 0.210 1.220 ± 0.120 60 0.643 ± 0.076 0.110 ± 0.015180 0.198 ± 0.026 0.038 ± 0.005 570 0.062 ± 0.004 0.020 ± 0.001 10200.030 ± 0.003 N/A

1. A method for detecting the presence of a disease in a biologicaltissue which may contain said disease, wherein said disease ischaracterized by the expression of tenascin-C in said tissue and whereinsaid disease is selected from the group consisting of cancer, psoriasis,and atherosclerosis, the method comprising: a) attaching a marker thatcan be used in in vivo diagnostics to a tenascin-C nucleic acid ligandto form a marker-nucleic acid ligand complex wherein said tenascin-Cnucleic acid ligand is selected from the group consisting of SEQ ID NO:4–65; b) exposing said biological tissue which may contain said diseaseto said marker-nucleic acid ligand complex; and c) detecting thepresence of said disease in said tissue by detecting the presence ofsaid marker-nucleic acid ligand in said tissue.
 2. The method of 1wherein said marker is selected from from the group consisting ofradionuclides, fluorophores, magnetic compounds, and biotin.
 3. Themethod of 2 wherein said radionuclide is selected from the groupconsisting of technetium-99m (Tc-99m), Re-188, Cu-64, Cu-67, F-18, ¹²⁵I,¹³¹I, ¹¹¹In, ³²P, and ¹⁸⁶Re.
 4. The method of 3 wherein said marker istechnetium-99m.
 5. The method of 4 wherein said tenascin-C nucleic acidligand comprises a linker.
 6. The method of 5 wherein said linker is(CH₂CH₂O)₆.
 7. The method of 5, wherein said linker has the structure


8. The method of 1 wherein said tenascin-C nucleic acid ligand is5′-B-G667667CG-(CH₂CH₂O)₆-CGUCGCCGU77U667U6UUUU6CUCCCU65 wherein: allpyrimidines are 2′F; 6=2′OMe G; 7=2′OMe A; 5=3′—3′dT; and B=linker. 9.The method of 8 wherein said technetium-99m is associated with achelator.
 10. The method of 9, wherein said complex is


11. The method of 10 wherein said complex is


12. The method of 1 further comprising attaching a therapeutic ordiagnostic agent to said complex.
 13. The method of 1 wherein saiddisease is cancer.
 14. The method of 1 wherein said tenascin-C nucleicacid ligand is identified by: i) contacting a candidate mixture ofnucleic acids with tenascin-C wherein nucleic acids having an increasedaffinity to tenascin-C relative to the candidate mixture may bepartitioned from the remainder of the candidate mixture; ii)partitioning the increased affinity nucleic acids from the remainder ofthe candidate mixture; iii) amplifying the increased affinity nucleicacids to yield a mixture of nucleic acids with relatively higheraffinity and specificity for binding to tenascin-C, whereby a nucleicacid ligand of tenascin-C is identified.